1.8.5 Micro RNA

Figure 1.8.12 General cloverleaf secondary  Mitochondria and chloroplasts contain distinctive, structure of tRNAs. The large dots on the somewhat smaller tRNAs. Cells have at least one kind of backbone represent nucleotide residues; the lines represent base pairs. Transfer RNAs vary in length from 73 to 93 nucleotides. Extra tRNA for each amino acid; at least 32 tRNAs are required to recognize all the amino acid codons (some recognize nucleotides occur in the extra arm or in the D more than one codon), but some cells use more than 32. arm. At the end of the anticodon arm is the  Yeast alanine tRNA (tRNAAla), the first nucleic acid to anticodon loop, which always contains seven be completely sequenced (Fig.1.8.11), contains 76 unpaired nucleotides. The D arm contains two nucleotide residues, 10 of which have modified bases. or three D (5,6-dihydrouridine) residues, Comparisons of tRNAs from various species have depending on the tRNA. In some tRNAs, the D revealed many common denominators of structure arm has only three hydrogen-bonded base (Fig.1.8.12). pairs. In addition to the symbols explained in  Eight or more of the nucleotide residues have modified

Figure 1.8.11 : Pu, purine nucleotide; Py, bases and sugars, many of which are methylated pyrimidine nucleotide; G*, guanylate or 2’-O- derivatives of the principal bases. Most tRNAs have amethylguanylate guanylate (pG) residue at the 5’ end, and all have the trinucleotide sequence CCA at the 3’ end.  When drawn in two dimensions, the hydrogen-bonding pattern of all tRNAs forms a cloverleaf structure with four arms; the longer tRNAs have a short fifth arm, or extra arm (Fig. 1.8.12). In three dimensions, a tRNA has the form of a twisted L (Fig.1.8.13 ).

1.8.5 Micro RNA

Small ribonucleic acid (RNA) can act as a specific regulator of gene expression. This discovery has been an exciting breakthrough in Biological Sciences of the past decade, culminating in last year’s Nobel Prize in Physiology or Medicine awarded to Andrew Fire and Craig Mello. They discovered that exogenous doublestranded RNA can be used to specifically interfere with gene function. This phenomenon was called RNA interference (RNAi). They also speculated that organisms might use double-stranded RNA naturally as a way of silencing genes. It was then shown that RNA interference was mediated by 22 nucleotide single-stranded RNAs termed small interfering RNAs (siRNAs) derived from the longer double-stranded RNA precursors. The small interfering RNAs were found to repress genes by eliminating the corresponding messenger RNA transcripts, and thus, preventing protein synthesis.  Several hundred genes in our genome encode small functional RNA molecules collectively called microRNAs (miRNAs).  Precursors of these miRNA molecules form structures of double-stranded RNA that can activate the RNA interference machinery.

Figure 1.8.13 Three-dimensional structure of yeast tRNAPhe deduced from x-ray diffraction analysis. The shape resembles a twisted L.

Unit 1  MicroRNAs down regulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation.  The first miRNA was discovered in 1993 by Victor Ambros and colleagues Rosalind Lee and Rhonda Feinbaum  A genetic screen in the roundworm Caenorhabditis elegans, a millimetre-long animal used as a model organism in biological research, identified genes involved in developmental timing  Surprisingly, one of the genes, termed lin-4, did not encode a protein but instead a novel 22nucleotide small RNA. Seven years later, it was discovered that a second 22-nucleotide small RNA of this type, let-7, a gene also involved in C. elegans developmental timing.  The lin-4 and let-7 small regulatory RNAs soon became very exciting for two reasons. Firstly, homologs of the let-7 gene were identified in other animals including humans  The conservation of let-7 across species suggested an important and fundamental biological role for this small RNA. Secondly, the mechanism of RNA interference (RNAi) was discovered at that time, and it became clear that miRNA and RNAi pathways were intricately linked and shared common Figure 1.8.14 The biogenesis and function of miRNAs. a Primary components. miRNAs (pri-miRNA) are transcribed from longer encoding DNA  These small non-coding RNAs were named microRNAs (miRNAs)  Subsequently, many more short sequences (miRNA genes). The pri-miRNA contains one or more stem-loop structures of about 70 bases. In the nucleus, the ribonuclease enzyme Drosha excises the stem-loop structure to form the precursor miRNA (pre-miRNA). b After export into the cytoplasm, the pre-miRNA is cleaved by the ribonuclease Dicer to regulatory RNAs were identified generate a short RNA duplex (miRNA:miRNA*). c The mature in almost all multicellular single-stranded miRNA is incorporated into the RNA-induced organisms, including flowering silencing complex (RISC), while the complementary strand plants, worms, flies, fish, frogs, (miRNA*) is usually rapidly degraded. The miRNA incorporated mammals into the silencing complex can bind to the target messenger RNA  To date, more than 500 human by base pairing, causing inhibition of protein translation and/or miRNAs have been degradation of the target messenger RNA experimentally identified.  This makes miRNAs one of the most abundant classes of regulatory genes in humans. MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation.  MicroRNAs are sequentially processed from longer precursor molecules that are encoded by the miRNA (Fig. 1.8.14).  MiRNA genes are referred to by the same name (termed mir) written in italics to distinguish them from the corresponding mature miRNA (termed miR) followed by a number, e.g., mir-1 or miR-1. The encoding DNA sequence is much longer than the mature miRNA. Two ribonuclease enzymes, Drosha and Dicer, subsequently process the primary transcripts (or pri-miRNA) to generate mature miRNAs.

The primary transcripts contain one or more stem-loop structures of about 70 bases. Stem-loops are double-stranded RNA structures consisting of a nucleotide sequence that can fold back on itself to form a double helix with a region of imperfect base pairing that forms an open loop at the end  The ribonuclease Drosha excises the stem-loop structure to form the precursor miRNA (or pre-miRNA) After export into the cytoplasm, the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex. After untwisting, one

RNA strand becomes the mature singlestranded miRNA, while the complementary strand, termed miRNA, is usually rapidly degraded  MicroRNAs recognize their targets based on sequence complementarity. The mature miRNA is partially complementary to one or more messenger RNAs. In humans, the complementary sites are usually within the 3′-untranslated region of the target messenger RNA.  To become effective, the mature miRNA forms a complex with proteins, termed the

RNA-induced silencing complex. The miRNA incorporated into the silencing complex can bind to the target messenger

RNA by base pairing.  This base pairing subsequently causes inhibition of protein translation and/or degradation of the messenger RNA (Fig. 1.8.14c).  Protein levels of the target gene are consequently reduced, whereas messenger RNA levels may or may not be decreased. In humans, miRNAs mainly inhibit protein translation of their target genes and only infrequently cause degradation or cleavage of the messenger

RNA  The biological role and in vivo functions of most mammalian miRNAs are still poorly understood. In invertebrates, miRNAs regulate developmental timing (e.g., lin-4), neuronal differentiation, cell proliferation, growth control, and programmed cell death  In mammals, miRNAs have been found to play a role in embryogenesis and stem cell maintenance , hematopoietic cell differentiation and brain  To date, knowledge of human miRNAs has been primarily descriptive. MicroRNA expression has been found to be deregulated in a wide range of human diseases including cancer. However, it remains uncertain whether altered miRNA expression is a cause or consequence of pathological processes. The underlying mechanisms of why and how miRNAs become deregulated are largely unknown. Although bioinformatics approaches can predict thousands of genes that are potentially targeted and regulated by miRNAs based on sequence complementarity, only very few miRNA target genes have been functionally validated.

Figure 1.8.15 Model for miRNA translational repression and RNA interference mediated by the RNA-induced interfering complex (RISC). Processing of both miRNA precursors into miRNAs and long double-stranded RNAs into short interfering RNAs (siRNAs) requires the Dicer ribonuclease. In both cases, cleavage by Dicer yields a double-stranded RNA intermediate containing 21–23 nucleotides per strand with twonucleotide 3’ single-strand ed tails. One strand of this intermediate assembles with multiple proteins to form an RNA induced silencing complex (RISC). In siRNA function, the target mRNA hybridizes perfectly with the RISC RNA, leading to cleavage of the target. In miRNA function, the RISC RNA forms a hybrid with the target mRNA that contains some base-pair mismatches; in this case translation of the target mRNA is blocked. This is the situation for the lin-4 and let-7 miRNAs in C. elegans. [Adapted from G. Hutvagner and P. D. Zamore, 2002, Science 297:2056; see also V. Ambrose, 2001, Cell 107:823.]

miRNA and siRNA can be used to treat inherited genetic disorders, cancer, obesity …etc.

Critical thinking Questions 1. How does cordycepin (3’-deoxyadenosine) block the synthesis of RNA? 2. A negatively supercoiled DNA molecule undergoes a B to Z transition over a segment of 360 base pairs. What is the effect on the writhing (supercoiling)? 3. Why is HAP column used to distinguish single stranded and double stranded DNA? 4. To precipitate DNA, an alcohol like ethanol or propanol is added to an aqueous DNA solution. Why should Na+ or NH4+ also be added? 5. A 41.5 nm-long duplex DNA molecule in the B-conformation adopts the A conformation upon dehydration How long is it now? What is its approximate number of base pairs?